β-coronaviruses reshape host cell endomembranes to form double-membrane vesicles (DMVs) for genome replication and transcription. Ectopically expressed viral nonstructural proteins nsp3 and nsp4 interact to zipper and bend the ER for DMV biogenesis. Genome-wide screens revealed the autophagy proteins VMP1 and TMEM41B as important host factors for SARS-CoV-2 infection. Here, we demonstrated that DMV biogenesis, induced by virus infection or expression of nsp3/4, is impaired in the VMP1 KO or TMEM41B KO cells. In VMP1 KO cells, the nsp3/4 complex forms normally, but the zippered ER fails to close into DMVs. In TMEM41B KO cells, the nsp3–nsp4 interaction is reduced and DMV formation is suppressed. Thus, VMP1 and TMEM41B function at different steps during DMV formation. VMP1 was shown to regulate cross-membrane phosphatidylserine (PS) distribution. Inhibiting PS synthesis partially rescues the DMV defects in VMP1 KO cells, suggesting that PS participates in DMV formation. We provide molecular insights into the collaboration of host factors with viral proteins to remodel host organelles.

中文翻译：

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VMP1和TMEM41B对于β-冠状病毒感染期间DMV的形成至关重要

β-冠状病毒重塑宿主细胞内膜，形成双膜囊泡（DMV），用于基因组复制和转录。异位表达的病毒非结构蛋白 nsp3 和 nsp4 与拉链相互作用并弯曲内质网以实现 DMV 生物发生。全基因组筛选显示自噬蛋白 VMP1 和 TMEM41B 是 SARS-CoV-2 感染的重要宿主因子。在这里，我们证明了由病毒感染或 nsp3/4 表达诱导的 DMV 生物发生在 VMP1 KO 或 TMEM41B KO 细胞中受损。在 VMP1 KO 细胞中，nsp3/4 复合物正常形成，但带拉链的 ER 无法闭合至 DMV。在 TMEM41B KO 细胞中，nsp3-nsp4 相互作用减少，DMV 形成受到抑制。因此，VMP1 和 TMEM41B 在 DMV 形成过程中的不同步骤发挥作用。 VMP1 可调节跨膜磷脂酰丝氨酸 (PS) 分布。抑制PS合成可部分挽救VMP1 KO细胞中的DMV缺陷，表明PS参与DMV形成。我们提供关于宿主因子与病毒蛋白相互作用以重塑宿主细胞器的分子见解。